Apparatus for stimulating an animal cell and recording its physiological signal and production and use methods thereof

ABSTRACT

The present invention provides an apparatus for stimulating an animal cell and recording its physiological signal and methods of making and using thereof. The purpose of the present invention is to provide an apparatus for stimulating an animal cell and recording its physiological signal that is efficient, convenient, and accurate. The apparatus for stimulating an animal cell and recording its physiological signal of the present invention comprises a poor conductive substrate, wherein on at least one surface of the substrate is provided at least one unit conductive polymer layer and at least one good conductive microelectrode.

FIELD OF INVENTION

The present invention relates to an apparatus for stimulating,particularly electrically stimulating, an animal cell and recording itsphysiological signals, as well as methods of making and using suchapparatus.

BACKGROUND OF THE INVENTION

The ability of controlling neuronal growth and differentiation will beuseful for the study of the underlying mechanisms of the nervous system,the treatment of neuronal diseases, and the effective repair of damagesto nerve tissues. It has therefore been the focus of scientific researchfor a long time.

After years of study, it was realized that electrical stimulation canenhance neurite outgrowth in vitro and enhance nerve cell regenerationin vivo. Work by other researchers has indicated that neurite outgrowthwas enhanced on the surface of piezoelectric material (Aebischer, etal., Piezoelectric guidance channels enhance regeneration in the mousesciatic nerve after axotomy, Brain Research, 1987, 436(1), 165-168).This effect has been attributed to the presence of surface-bound chargesresulting from minute mechanical stresses on the material. The exactmechanism of the observation is not yet clear. One theory is thatcertain proteins or other molecules that are critical to neuriteextension become redistributed in the electrical field. Alternatively,these proteins could have undergone conformational changes that arefavorable to neurite extension.

Conductive polymers represent a new class of materials whose electricaland optical properties can be controllably varied over a wide range,often in a reversible manner. Conductive polymers are stable, can beused in physiological cell culture media or body fluid for extendedtime, and have good compatibility with neurons. (C. E. Schmidt, V. R.Shastri, J. P. Vacanti, R. Langer, Stimulation of neurite outgrowthusing an electrically conducting polymer, Proc. Natl. Acad. Sci. USA,1997, 94, 8948-8953). It was also shown recently that electricalstimulation of neurons using conductive polymers as conductive mediaenhances neurite outgrowth. (A. Kotwal, C. E. Schmidt, Electricalstimulation alters protein adsorption and nerve cell interactions withelectrically conducting biomaterials, Biomaterials, 2001, 22, 1055-1064;C. E. Schmidt, V. R. Shastri, J. P. Vacanti, R. Langer, Stimulation ofneurite outgrowth using an electrically conducting polymer, Proc. Natl.Acad. Sci. USA, 1997, 94, 8948-8953). Researchers have also implantedconductive polymers as a scaffold in vivo, and used electrical stimulusto connect severed nerve tissues under the guidance of the scaffold (V.R. Shastri, C. E. Schmidt, R. S. Langer, J. P. Vacanti, U.S. Pat. No.6,095,148, Aug. 1, 2000). Use of microelectrodes or arrays ofmicroelectrodes to record electrophysiological signals of neurons andnerve networks has been studied since the 1970's, and tremendousprogress has been made in recent years. One way is to insert multiplemicroelectrodes into a live subject to measure extracellular signals.The electrodes can be in the shape of spikes, with multiple electrodesbeing a cluster of spikes (E. Fernandez, J. M. Ferrandez, J.Ammermuller, R. A. Normann, Population coding in spike trains ofsimultaneously recorded retinal ganglion cells, Brain Research, 2000,887, 222-229; D. J. Warren, E. Fernandez, R. A. Normann, High resolutiontwo-dimensional spatial mapping of cat striate cortex using a100-microeletrode array, Neuroscience, 2001, 105(1), 19-31; P. J.Rousche, R. S. Petersen, S. Battiston, S. Giannotta, M. E. Diamond,Examination of the spatial and temporal distribution of sensory corticalactivity using a 100-electrode array, Journal of Neuroscience Methods,1999, 90, 57-66), or can be positioned in a cone, with each electrodebeing a planar electrode on the bottom surface of the cone (G. Ensell,D. J. Banks, P. R. Richards, W. Balachandran, D. J. Ewins, Silicon-basedmicroelectrodes for neurophysiology, micromachined formsilicon-on-insulator wafers, Medical & Biological Engineering &Computing, 2000, 38, 175-179); another method is to use atwo-dimensional array of microelectrodes to simultaneously measuremultiple measured cells cultured in vitro (Y. Jimbo, A. Kawana, P.Parodi, V. Torre, The dynamics of a neuronal culture of dissociatedcortical neurons of neonatal rats, Biological Cybernetics, 2000, 83,1-20; T. Tateno, Y. Jimbo, Activity-dependent enhancement in thereliability of correlated spike timings in cultured cortical neurons,Biological Cybernetics, 1999, 80, 45-55; M. P. Maher, J. Pine, J.Wright, Y. C. Tai, The neurochip: a new multielectrode device forstimulating and recording from cultured neuron, Journal of NeuroscienceMethods, 1999, 87, 45-56); the third method is to make spikes containingthe microelectrode arrays of the second method along with embryonicneurons, insert the spikes into a live subject, and observe theintegration and communication of signals between cells on themicroelectrode array and cells in the subject. (J. Pine, M. Maher, S.Potter, Y. C. Tai, S, Tatic-Lucic, J. Wright, A cultured neuron probe,Proceedings of IEEE-EMBS Annual Meeting, Amersterdam, the Netherlands,1996, November, paper #421). The advantage of measuring signals in vitrois that one can control the positioning of the cell and the condition ofthe cell culture, thereby studying various functions of the neuronsclearly and conveniently. However, the direction of neurite outgrowth isusually random and hard to control, which makes it difficult toestablish a nerve network for the purpose of recording the communicationof electrophysiological signals. It also makes it difficult forimplanted neurons to integrate into the nervous system of the subjectand communicate with the nervous system. Some researchers havemechanically forced neurons to grow only in defined channels. This kindof mechanical restriction, however, affects the normal outgrowth ofneurites. Other researchers have used patterns formed by materials suchas metal oxide to study the guidance of these materials on neurites(Yashihiko Jimbo, P. C. Robinson, Akio Kawana, Simultaneous Measurementof Intracellular Calcium and Electrical Activity from Patterned NeuralNetworks in Culture, IEEE transaction on biomedical engineering, 1993,40(8), 804-810).

DETAILED DESCRIPTION OF THE INVENTION

The purpose of the present invention is to provide an efficient,convenient, and accurate apparatus for stimulating an animal cell andrecording its physiological signal. To achieve such purpose, the presentinvention provides an apparatus for stimulating an animal cell andrecording its physiological signal, such apparatus comprising asubstrate comprising a poor conductive material, wherein on at least oneface of the substrate is provided at least one unit conductive polymerlayer and at least one good conductive microelectrode.

To prevent the conductive polymer from detaching from the substrate asresult of extended exposure to culture media or body fluid, anintermediate layer between the substrate and the conductive polymerlayer can be provided. Such intermediate layer can be made of gold orplatinum. One function of the intermediate layer is to serve as acarrier for electrical polymerization. Another function is to allowconductive polymer to adhere strongly.

The substrate can be a two-dimensional planar structure; or it cancontain one or more wells for positioning cells.

The material used to make the substrate can be rigid or flexible, andcan be chosen from silicon, glass, polymer, or metal oxides.

The conductive polymer serves to guide the outgrowth of the neurites.Accordingly, the conductive polymers can form a grid-like pattern withdisconnected junctions, with nerve cells at the junctions. The neuriteswill therefore grow along the conductive polymer pattern and communicatewith each other. Because the diameters of neurites are usually 1-2microns, the width of each band of conductive polymers is usuallysmaller than 5 microns. If the band is too narrow or too broad, theability of the conductive polymers to guide the neurites properly willbe affected. The pattern of the conductive polymers should be chosen ina way that facilitates the formation of nerve networks among theneurites.

The conductive polymer described herein can comprise multiple units.These units can be interconnected and share a single pair of stimulatingelectrodes. Alternatively, the units may not be connected with eachother, each unit having their own pair of stimulating electrodes. If theunits are not interconnected, the stimulating parameters (such ascurrent intensity, duration, amplitude, and frequency) for eachconductive polymer unit can be either the same or different.

The thickness of the conductive polymer layer described herein can beuniform or not uniform. The thickness of the conductive polymer layercan be between several nanometers to several millimeters. When the cellsare to be observed in vitro under an inverse microscope, the thicknessof the conductive polymer layer is preferably below 500 nanometers. Thisprovides high light permeability and allows the polymers to properlyadhere to the substrate.

The material that the conductive polymer can be made of includespolyaniline, polypyrrole, polythiophene or their derivatives,copolymers, or mixtures thereof. The conductivity of the conductivepolymer can be adjusted according to spatial and temporal requirements.When microelectrodes are used to record electrophysiological signals,the conductive polymer layer can either be adjusted to be nonconductiveor remain being conductive, as long as the measurement of theelectrophysical signals is not affected.

The microelectrodes described herein may comprise more than onemicroelectrodes, which can be arranged into microelectrode arrays. Thematerial from which the microelectrodes are made can be gold, platinum,or indium-tin oxide (ITO).

The conductive polymer layer and the microelectrodes described hereincan be connected or not connected to each other. When not connected, theconductive polymer layer and microelectrodes can be about 1 to about 50microns apart. The distance between conductive polymer andmicroelectrode in each unit can be the same or different.

To prevent the escape of cultured cells, the area above the substratethat is not covered by conductive polymers and is not the area for cellgrowth or microelectrode measurement can be coated with insulationmaterials. The insulation material can be made of nonconductivematerials (such as polyimide or other materials with goodbiocompatibility) that are not toxic to cells. The thickness of theinsulation layer is usually 5 to 100 microns.

Another purpose of the present invention is to provide a simple methodof making the apparatus for stimulating animal cells and recording thephysiological signals described above.

In one embodiment, the method of making an apparatus for stimulatinganimal cells and recording the physiological signals comprises the stepsof: (a) depositing good conductive microelectrodes and their connectingwires on a substrate through evaporation or sputter; (b) depositing atleast one unit of conductive polymer layer with a desired pattern on thesubstrate by PVD, CVD, electrical polymerization, or macromoleculeself-assembly; and (c) depositing some metallic wires underneath theconductive polymer layer for connecting the conductive polymers to thestimulating electrodes.

Another purpose of the present invention is to provide a method of usingan apparatus for stimulating animal cells and recording physiologicalsignals.

In one embodiment, the method of using an apparatus for stimulatinganimal cells and recording physiological signals comprises the steps of:(a) passing electrical current through conductive polymers eithercontinuously or intermittently, wherein the electrical stimulation canbe direct current or alternating current, wherein the electric currentintensity can be of pA to mA scale, and wherein for alternating currentstimulation, the stimulation frequency can be 1-10⁶ Hz; and (b)recording physiological signals generated from cells being testedthrough the microelectrodes by measuring electrical current, electricalpotential, or impedance electrical signals.

The apparatus for stimulating animal cells and recording physiologicalsignals described herein can be two dimensional squares, circles, or anyother irregular shapes. In other embodiments, the apparatus is athree-dimensional hollow cylinder, a globe, a cubic, a cuboid, a cone,or any other irregular shapes.

The present invention is further illustrated through the followingdescription of the figures and example.

DESCRIPTION OF THE FIGURES

FIG. 1 provides a structural diagram of a subregion of an apparatus ofthe present invention.

FIG. 2 provides an overview of an apparatus of the present invention.

FIG. 3 provides a magnified view of microelectrode arrays.

FIG. 4 provides a sectional view of a single culture unit used forstudying nerve networks in vitro.

FIG. 5 provides a three dimensional diagram of an apparatus of thepresent invention that is used for implanting into a live subject.

EXAMPLES Example 1 Making of the Apparatus of the Prevent Invention

On one surface of a 350 micron-thick square glass substrate, use routinephotoetching method to produce a 300-nm thick layer of gold, withmeasuring microelectrodes, stimulating electrodes, and connecting wireswith a desired pattern.

Apply electricity to the stimulating electrodes. Use electrochemicalpolymerization methods to form a 100 nm-thick layer of polyanilineconductive polymer on top of the gold layer that is connected to thestimulating electrodes, forming the desired pattern.

Use spin-coating method to create an insulation layer on the top. Usephotoetching method to generate the desired structure.

An apparatus for stimulating animal cells and recording itsphysiological signals can be made using the above method.

In this example, the substrate can be chosen from a wide range ofmaterials, as long as it is a poor conductive material. The thicknessand shape of the substrate can be chosen according to the needs of theexperiment. To make efficient use of the space, more than one surface ofthe substrate can be utilized as described above. The electrode layercan be made of platinum, ITO, or other kinds of good electricallyconductive materials. The thickness of the electrode layer is preferablybetween 30 nm to 400 nm. The material from which the conductive polymersare made of can be chosen from a wide variety of materials, and dependson the needs of the experiment. For example, the material can bepolyaniline, polypyrrole, polythiophene, or their derivatives,copolymers, or mixtures thereof. Other methods of coating orphotoetching can also be used to generate electrodes and conductivepolymer layers and to form desired patterns.

Example 2 Structure of the Present Invention

As shown in FIGS. 1, 2, and 3, the apparatus for stimulating animalcells and recording its physiological signals comprises the 400micron-thick square silicon substrate 4; the 100-micron thickpolypyrrole conductive polymer layer 3 on top of substrate 4, theconductive polymer layer being connected to two electrodes (notindicated in this figure); the sixteen 40 nm-thick platinummicroelectrodes 2 on top of substrate 4, each microelectrode having anoutput electrode (not indicated in this figure). There is a 25-microngap between the conductive polymer 3 and the microelectrode 2. Areas onsubstrate 4 that are not covered by conductive polymers 3 and are notareas for cell growth or microelectrodes measurement are covered by apolyimide insulation layer 1. The insulation layer has a thickness of 10microns, forming a 3-7 micron-wide channel that prevents cells fromescaping.

As shown in FIG. 2, there is a small cell culture chamber 5 on top ofthe substrate for holding cell culture media. The chamber is made ofbiocompatible materials such as polyimide. The height of the culturechamber 5 is usually bigger than 100 mm, and the area of the chamberdepends on the number and distribution of the microelectrodes in themicroelectrode array.

In this example, there is an intermediate layer between the substrateand the conductive polymer layer (not shown in the figure) forpreventing the conductive polymer layer from detaching from thesubstrate as a result of extended exposure to the culture medium or bodyfluid.

In the present example, the size and shape of the apparatus, as well asthe choice of the surface or surfaces of the substrate for depositingconductive polymer layers or microelectrodes, are determined by theneeds of the experiment. As shown in FIG. 5, to facilitate the use ofthe apparatus in vivo, the two lateral sides of the substrate 4 are bothcovered with conductive polymer layer 3 and microelectrodes 2. Neuronscan be placed on both sides of the substrate, thus to increase celldensity and enhance efficiency. As shown in FIG. 5, the apparatus forimplanting in vivo contains a top layer 6. This layer is useful forpreventing cell damages during implanting and preventing cells fromescaping in the live subject. The material from which layer 6 is made ofis preferably biocompatible, stable, and nonconductive (such aspolyimide).

Example 3 Use of the Apparatus of the Present Invention to Study NerveNetwork In Vitro

Place neuron 7 on the apparatus as shown in FIG. 4. Introduce electricalcurrent intermittently through the conductive polymer. The electricalstimulation can be direct current or alternating current, and thestimulation frequency can be 1-10⁶ Hz. The physiological signals of thecell being studied are measured through microelectrode 2.

After the intermittent electrical stimulation (constant voltage at 100mV), and culturing of the cells for several days, the growth of thestimulated cells, as measured by parameters such as the size of the cellbody, the length of the neurites, is measured and compared with those ofunstimulated cells. In addition, cells are stimulated by electrical orchemical signals, and the response of the cells to the variousstimulations is monitored through the microelectrodes, by measuring thechanges of the electrical potential on the cell membrane.

Example 4 Use of the Apparatus to Study Nerve Network In Vivo

Implant the apparatus as shown in FIG. 5 into a live subject. Theneuronal network on the apparatus is used to connect damaged nervefibers in the subject. The apparatus is also used to measure theresponse of neurons in local nerve tissues to external stimuli(including electrical and medicinal stimuli). Furthermore, the apparatuscan be used to electrically stimulate local nerve tissues, therebystimulate the repair and regeneration of damaged nerves.

Industrial Utility

The present invention applies conductive polymer and microelectrodefabrication technologies to neural science, and utilizes electricalstimulation through conductive polymers to effectively control the speedand direction of neurite outgrowth. This allows effective recording ofelectrophysiological signals of nerve network in vitro, and makes itpossible to further study signal transduction and underlying mechanismsof the nervous system. The present invention also provides animplantable apparatus that allows the implantation of nerve cells invivo, integration of implanted nerve cells with the main nerve system,and repair of damaged nerve tissues. The apparatus therefore hasimportant medical application values. It is also expected that theapparatus will be useful for the treatment of urine incontinence, retinainjury, or other neuronal diseases.

The apparatus of the present invention is not only useful for the studyof different kinds of cells derived from various animal nervous systems,but also useful for the study of cells derived from non-neuronal cells.

1. An apparatus for stimulating an animal cell and recording itsphysiological signals, such apparatus comprising a poor conductivesubstrate, wherein on at least one surface of the substrate is providedat least one unit conductive polymer layer and at least one goodconductive microelectrode.
 2. An apparatus for stimulating an animalcell and recording its physiological signals according to claim 1,further comprising an intermediate layer between the substrate and theconductive polymer layer.
 3. An apparatus for stimulating an animal celland recording its physiological signals according to claim 2, whereinthe intermediate layer is gold or platinum.
 4. An apparatus forstimulating an animal cell and recording its physiological signalsaccording to claim 1, wherein the substrate is a two-dimensional planarstructure.
 5. An apparatus for stimulating an animal cell and recordingits physiological signals according to claim 1, wherein the substratecomprises at least one well for positioning the cells.
 6. An apparatusfor stimulating an animal cell and recording its physiological signalsaccording to claims 1, 2, 3, 4, or 5, wherein the conductive polymerlayer has a grid-like pattern with disconnected junctions.
 7. Anapparatus for stimulating an animal cell and recording its physiologicalsignals according to claims 1, 2, 3, 4, or 5, wherein the conductivepolymer layer comprises multiple units that are connected to each otherand share a single pair of stimulating electrodes.
 8. An apparatus forstimulating an animal cell and recording its physiological signalsaccording to claims 1, 2, 3, 4, or 5, wherein the conductive polymerlayer comprises multiple units that are not connected to each other,each unit comprising a pair of stimulating electrodes.
 9. An apparatusfor stimulating an animal cell and recording its physiological signalsaccording to claim 1, wherein the conductive polymer layer is made ofpolyaniline, polypyrrole, polythiophene or their derivatives,copolymers, or mixtures thereof.
 10. An apparatus for stimulating ananimal cell and recording its physiological signals according to claim1, said apparatus comprising more than one microelectrodes, wherein saidmicroelectrodes are arranged in a microelectrode array.
 11. An apparatusfor stimulating an animal cell and recording its physiological signalsaccording to claim 1, wherein the material for making the microelectrodeis gold, platinum, or ITO.
 12. An apparatus for stimulating an animalcell and recording its physiological signals according to claim 1,wherein the conductive polymer is connected with the microelectrode. 13.An apparatus for stimulating an animal cell and recording itsphysiological signals according to claim 1, wherein the conductivepolymer and the microelectrode is 1-50 microns apart.
 14. An apparatusfor stimulating an animal cell and recording its physiological signalsaccording to claim 1, wherein the area above the substrate that is notcovered by conductive polymers, and is not the area for cell growth ormicroelectrode measurement, is coated with an insulation material layer.15. A method of making an apparatus for stimulating an animal cell andrecording its physiological signals, comprising: (a) depositing goodconductive microelectrodes and their connecting wires on a substratethrough evaporation or sputter; (b) depositing at least one unit ofconductive polymer layer with a desired pattern on the substrate by PVD,CVD, electrical polymerization, or macromolecule self-assembly; and (c)depositing some metallic wires underneath the conductive polymer layerfor connecting the conductive polymers to stimulating electrodes.
 16. Amethod according to claim 15, further comprising the step of depositingan insulation layer on the apparatus using photoetching to form adesired structure.
 17. A method according to claim 15, furthercomprising the step of depositing an insulation layer with desiredpattern by photoetching, wherein said step occurs before the step ofdepositing the conductive polymer layer.
 18. A method of using anapparatus for stimulating an animal cell and recording its physiologicalsignals, comprising the steps of (a) passing electrical current throughconductive polymers either continuously or intermittently, wherein theelectrical stimulation is direct current or alternating current, whereinthe electric current intensity is of pA to mA scale, and wherein foralternating current stimulation, the stimulation frequency is 1-10⁶ Hz;and (b) recording physiological signals generated from cells beingtested through the microelectrodes by measuring the electrical current,electrical potential, or impedance electrical signals.